Project Summary/Abstract Recently therapies targeting the immune response, such as PD1 antagonists, have demonstrated unprecedented success in the treatment of metastatic melanoma. However, with the majority of patients failing to respond, there is a need for an understanding of the mechanisms of resistance and novel approaches to improve immunotherapies. This proposal seeks to address that need by investigating the roles of IL-10 in patient response to immunotherapy. Preliminary data demonstrate that patients responding to PD1 blockade had increased T-cell STAT3 and IL10 expression, which was absent in non-responding patients. In vitro, treatment of T-cell cultures with ?PD1 resulted in STAT3 dependent increases in IL-10 production. Treatment of T-cells with exogenous IL-10 enhanced T-cell cytolytic functions, proliferation, memory and rescues anergic cells. These effects were enhanced when combined with ?PD1. However, IL10 treatment of total PBMC resulted in increased M2-like antigen presenting cells and myeloid derived suppressor cells, and in turn decreased T-cell effector functions. Based on these and published studies, we hypothesize that IL-10 induction is a biomarker of patient response to PD1 blockade therapy and that T-cell targeted delivery of IL-10 can enhance PD1 blockade immunotherapy. To address this hypothesis, the proposed research will investigate 1) changes in the production of IL-10 by different patient immune cell populations after PD1 blockade and the relation to patient outcome, 2) the mechanisms by which ?PD1 induces T-cell IL-10 production, 3) the mechanisms by which IL-10 has cytolytic promoting effects on T-cells and opposing effects in the presence of antigen presenting cells, 4) the ability of IL- 10 to enhance adoptive cell therapy in preclinical murine models, and 5) to test the efficacy of T-cell targeted delivery by an ?PD1/IL-10 conjugate to enhance immunotherapy. These lines of research will be addressed throughout the K99 phase of this grant and continue during the R00 phase. This research stands to have significant clinical impact through demonstrating novel mechanisms through which ?PD1 therapies function and improving patient responses by rationale combinations. In addition, this grant outlines my career development plan for obtaining the requisite training necessary to be a productive and successful independent academic researcher. This includes mentoring by Dr. Jeffrey Weber and Dr. Pratip Chattopadhyay and a Scientific Advisory & Career Development Committee consisting of Dr. Itai Yanai, Dr. Kwok-Kin Wong and Dr. James Mul. As part of my plan, I will take coursework in bioinformatics, grant writing and managing a lab. I will also train in techniques including single cell RNA-Seq, high dimension flow cytometery and murine models of adoptive cell therapy. The labs of Dr. Weber and Dr. Chattopadhyay and NYU Health will provide the resources critical to my training and to the proposed research, ensuring my success. Collectively, the proposed career development and research plans are expected to generate data with significant impact on the treatment of metastatic melanoma while propelling my career and setting the theme of my lab as an independent researcher.